Development of an in Vitro Aggregation Assay for Long Synthetic Polypeptide, Amyloidogenic Gelsolin Fragment AGElD187N 173–242
Laura Leimu, Oskar Haavisto, Victor Nesati, Patrik Holm, Antti Haapalinna, Rune Salbo, Ullamari Pesonen
Abstract
Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173–242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173–242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173–242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully.
Introduction
Gelsolin amyloidosis (AGel amyloidosis) is a hereditary systemic protein misfolding disease that is caused by mutations in the gelsolin gene. The functional mutations predispose the secreted plasma gelsolin to aberrant proteolytic cleavages. The first identified and best-known mutation is the D187N variant, the so-called Finnish variant [1]. This mutation compromises the stability of the protein and leads to the formation of amyloidogenic fragments. The main product is the 8 kDa fragment comprising of 70 amino acid residues, AGelD187N 173–242. The gelsolin amyloidosis patients with the Finnish mutation will experience a triad of ophthalmological, neurological and dermal symptoms as a cause of gelsolin fragment accumulation in the basement membrane of the skin, blood vessel walls, eyes and peripheral nervous system [2]. This will cause severe disease burden starting from young adulthood [3]. As currently the only treatments available are alleviation of symptoms and support care, there is a high unmet medical need for disease modifying treatments.
Materials and methods
Polypeptides
All synthetic polypeptides used in this study were supplied by Caslo ApS, Denmark. The full-length human plasma gelsolin (UniProtKB P06396-1) amino acid numbers, terminal modifications, reversed-phase-high performance liquid chromatography (RP-HPLC) purities and batch sizes are listed in Table 1. The used nomenclature is based on the recommendations by the International Society of Amyloidosis (ISA) nomenclature committee [25,26]. The name AGelD187N in the text refers to all three polypeptides used in the study and the name AGelD187N 173–242 is used interchangeably to refer to AGelD187N 173–242 (95%), which was mainly used in this study. The amino acid sequences of AGelD187N 173–242 and AGelD187N 173–243 are presented in Fig 1.
Results
Purity and N-terminal modification affect the aggregation tendency of synthetic AGelD187N
We investigated three different variants of the synthetic AGelD187N polypeptide in this study (Table 1). First two variants had different RP-HPLC purities (95% and 90%) and the third variant had an N-terminal acetylation, which is commonly added to synthetic peptides to enhance their biological activity. The N-terminally acetylated variant had 95% purity. All three polypeptides were sonicated in 6 M guanidine hydrochloride and monomerized by gel filtration chromatography. The monomerization yields of different AGelD187N polypeptides were from 10–30% (S1 Table). Immediately after monomerization, polypeptides were diluted and used at a 25 μM concentration in the ThT based aggregation assay. The large enhancement of its fluorescence emission upon fibril binding makes ThT an unusually sensitive and efficient reporter [28]. However, different fibril structures and morphologies may elicit different level of fluorescence intensity enhancement [28–31]. ThT binding and fluorescence may also be affected by selected assay conditions, additives and molecules that are tested as aggregation inhibitors [32–34].
Discussion
In this work we have described a practical and drug discovery suitable approach to study in vitro the aggregation kinetics of the main amyloidogenic polypeptide in Finnish gelsolin amyloidosis, the 70-residue gelsolin fragment AGelD187N 173–242. We have been able to show that synthetic AGelD187N 173–242, monomerized in a similar way to recombinant AGelD187N 173–242, is able to form fibrils typical of amyloid diseases. Reproducible aggregation kinetics are attainable with this full-length synthetic material when correctly handled. Earlier studies with shorter synthetic stretches of AGelD187N 173–242 are less in vivo relevant, since shorter peptides do not have all the features the disease-related full-length fragment has. On the other hand, the biotechnological production of all amyloidogenic polypeptides is cumbersome, and extremely pure recombinant material with sufficient yields are difficult to obtain. The utilization of synthetic material enables large screening campaigns where considerable amounts of amyloidogenic protein is needed.
Acknowledgments
Authors would like to thank Anja Vilkman for helping with the monomerization of AGelD187N 173-242. The work of LL was performed under the University of Turku Drug Research Doctoral Programme (DRDP). Electron microscopy samples were processed and analyzed in the Electron Microscopy Laboratory, Institute of Biomedicine, University of Turku.
Citation: Leimu L, Haavisto O, Nesati V, Holm P, Haapalinna A, Salbo R, et al. (2023) Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173–242. PLoS ONE 18(8): e0290179. https://doi.org/10.1371/journal.pone.0290179
Editor: Myriam M. Altamirano-Bustamante, Instituto Mexicano del Seguro Social, MEXICO
Received: April 11, 2023; Accepted: August 2, 2023; Published: August 17, 2023
Copyright: © 2023 Leimu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study has been conducted as a collaboration between Orion Pharma and University of Turku. During the study LL was employed by University of Turku in a collaboration project funded by Orion Pharma. The funder provided support in the form of salaries for authors VN, PH, AH and RS at the time of the study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: UP and OH declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. VN, PH, AH and RS were paid employees of Orion Pharma at the time of the study. Currently also LL is a paid employee of Orion Pharma. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0290179#abstract0
